Date of Award

5-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular and Systems Pharmacology

Research Advisor

Jun Yang, PhD

Committee

Taosheng Chen, PhD; Andrew M. Davidoff, MD; Kevin W. Freeman, PhD; Mark E. Hatley, PhD; Liqin Zhu, PhD

Keywords

Histone lysine demethylase, KDM4, mTOR, Neuroblastoma, Pediatric cancer, QC6352

Abstract

Neuroblastoma (NB) is a malignancy of improperly maturated nerve tissues of the sympathetic nervous system. It is the most diagnosed extracranial solid tumor of childhood, and it accounts for roughly up to 10% of all childhood malignancies and 15% of all childhood malignancy-related deaths. MYCN amplification, occurring in 20-25% of all NB patients and 50% of all high-risk NB patients, is the well-characterized genetic abnormality and is associated with ill-fated outcomes. Regrettably, in spite of the contemporary therapeutic advances and aggressive combinatorial treatments, the overall 5-year event-free survival rate is roughly 51% with a 5-year overall survival rate of nearly 63%. Unfortunately, close to 50% of high-risk NB patients develop disease relapses, which are largely incurable with no salvageable therapies so far. Recent evidence highlights that NB exhibits two cellular heterogeneities, namely adrenergic (ADRN) and mesenchymal (MES), which are tightly controlled by core regulatory circuitry-transcription factors (CRC-TFs). Driven by super-enhancers, these CRC-TFs, produce interrelated, feed-forward transcriptional loops that ultimately reinforce a specific gene expression program that defines the cellular state and identity of the cell. We showed that histone lysine demethylase subfamily 4 (KDM4) protein expression was higher in MYCN-amplified (MNA) NB cell lines and patient-derived xenografts (PDXs) compared with non-MNA counterparts. Additionally, genetic depletion of KDM4 correlated with reduced proliferation in vitro and in vivo. Interestingly, forced MYCN overexpression in MES-dominant NB cells facilitated MES to ADRN cellular state shift that was accompanied by induction of KDM4 proteins. Through various functional and genetic assays, we further validated the importance of the MYCN/KDM4/ADRN CRC-TF axis in maintaining the ADRN cellular state of NB. In view of these thrilling results, we opted to expand the clinical utility and examine the prospect of pharmacologic inhibition of KDM4 with QC6352, a selective and potent KDM4 inhibitor. Our data depicted that monotherapy QC6352 was selectively sensitive against MNA NB cells in vitro. Additionally, monotherapy QC6352 depicted satisfactory antitumor activities in high-risk cell line-based xenografts (NB1691), PDXs, and TH-MYCN/ALKF1178L transgenic mouse model in vivo. Molecularly, QC6352 reduced the mRNA and protein levels of oncogenic drivers and various ADRN CRC-TFs. Interestingly, QC6352 also decreased KDM4 expression at the translational protein level. Additionally, QC6352 culminated in favorable anticancer effects in vitro and in vivo, reflected by differentiation induction, apoptosis instigation, DNA damage, cell cycle cessation, and interestingly type-I interferon reactivation. Epigenomic-based investigations utilizing assay for transposase-accessible chromatin with sequencing (ATAC-seq) showed QC6352 reduced chromatin accessibility of MYCN and ADRN CRC-TFs, whereas chromatin immunoprecipitation with sequencing (ChIP-seq) and cleavage under targets and tagmentation (CUT&TAG) analyses showed KDM4 proteins were bound to genomic loci of MYCN and ADRN CRC-TFs, and QC6352 treatment promoted induction of the transcriptional repressive H3K9me3 mark. Lastly, combination therapy comprising QC6352 and standard-of-care chemotherapeutics (vincristine and irinotecan) led to 100% complete response in MNA NB xenografts without overt toxicity and accompanied by prolongation of survival. All in all, this research offers a solid proof-of-concept that pharmacologic inhibition of KDM4 with QC6352 may be translated from bench to bedside as a novel therapeutic tactic for high-risk MNA NB patients. Moreover, two orthogonal drug screens comprising CRISPR-Cas9 knockout (metabolism and kinase libraries), and high-throughput PRISM analysis highlighted enhanced anticancer effects between QC6352 and mammalian target of rapamycin (mTOR) inhibition. We demonstrated that combination of QC6352 and some mTOR inhibitors resulted in enhanced anticancer effects in vitro in several MNA and non-MNA NB cells. Functional genetic assays to validate the enhanced anticancer effects between dual KDM4 and mTOR inhibition failed. Lastly, combination of QC6352+rapamycin did not result in enhanced anticancer effects in SKNAS xenografts in vivo. Unless more in vivo work is performed, the present findings do not provide a compelling motivation to combine QC6352 with mTOR inhibition in the treatment of NB tumors.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0003-2286-2181

DOI

10.21007/etd.cghs.2023.0626

Additional Files
2023-013-Abuzaid-DOA.pdf (151 kB)
Declaration of Authorship
Supplemental Data for Chapter 3.pdf (2872 kB)
Supplemental Data for Chapter 3
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